Renal Denervation

Quantification of Renal Sympathetic Vasomotion as a Novel End Point for Renal Denervation Peter Ricci Pellegrino, Irving H. Zucker, Yiannis S. Chatzizisis, Han-Jun Wang, Alicia M. Schiller

Abstract—Renal sympathetic denervation, a potentially revolutionary interventional treatment for hypertension, faces an existential problem due to the inability to confirm successful ablation of the targeted renal sympathetic nerves. Based on the observation that renal sympathetic nerve activity exerts rhythmic, baroreflex-driven, and vasoconstrictive control of the renal vasculature, we developed a novel technique for identifying rhythmic sympathetic vascular control using a time- varying, 2-component Windkessel model of the renal circulation. This technology was tested in 2 different animal models of renal denervation; 10 rabbits underwent chronic, surgical renal denervation, and 9 pigs underwent acute, functional renal denervation via intrathecal administration of ropivacaine. Both methods of renal denervation reduced negative admittance gain, negative phase shift renal vascular control at known sympathetic vasomotor frequencies, consistent with a reduction in vasoconstrictive, baroreflex-driven renal sympathetic vasomotion. Classic measures like mean renal blood flow and mean renal vascular resistance were not significantly affected in either model of renal denervation. Renal sympathetic vasomotion monitoring could provide intraprocedural feedback for interventionists performing renal denervation and serve more broadly as a platform technology for the evaluation and treatment of diseases affecting the sympathetic nervous system. Graphic Abstract—A graphic abstract is available for this article. (Hypertension. 2020;76:1247-1255. DOI: 10.1161/ HYPERTENSIONAHA.120.15325.) • Data Supplement Key Words: baroreflex◼ heart failure ◼ homeostasis ◼ renal denervation ◼ sympathetic nervous system ◼ vascular resistance

Downloaded from http://ahajournals.org by on October 27, 2020 he sympathetic nervous system controls diverse aspects of Renal sympathetic outflow controls renin release, sodium Tbody homeostasis and plays a maladaptive role in hyper- reabsorption, and renal vascular tone.8 Renal sympathetic vas- tension,1 heart failure,2 and chronic kidney disease.3 Despite cular control represents an appealing end point for the assess- the importance of the sympathetic nervous system in health ment of sympathetic outflow for numerous reasons, including and disease, clinicians lack widely accessible measures of re- the availability of existing clinical technologies to measure gional sympathetic outflow. The inability to clinically assess blood flow in real-time and the potential generalizability of this master regulator negatively affects therapies targeting such a technique to other vascular beds. Renal vascular con- the sympathetic nervous system, acute monitoring of at-risk trol, however, is particularly complex, with the autoregulatory patients, and patient care for diseases involving the sympa- mechanisms of tubuloglomerular feedback and the myogenic 9 thetic nervous system. response exerting tight control over renal vascular resistance. For this reason, simple static measures like mean renal blood Nowhere is this more evident than for renal denervation, flow (RBF) and renal vascular resistance do not reliably re- a promising antihypertensive intervention that languished for flect sympathetic tone, and more sensitive, dynamic measures years after a pivotal clinical trial failed to show that renal de- of sympathetic vascular control are needed. nervation decreases blood pressure relative to a sham proce- In this study, we identify a baroreflex-driven rhythm in di- 4 dure. Although many explanations for this efficacy failure rect renal sympathetic nerve recordings from conscious rab- were advanced, convincing post hoc analysis of the trial in- bits, which corresponds to known key sympathetic vasomotor 5,6 dicated that the patients simply were not denervated, a fact frequencies in rabbits, pigs, and humans. Then, we lay out that was obscured to interventionists by the inability to assess a novel method for characterizing active rhythmic vascular the sympatholytic effect of these devices. With subsequent modulation with clear physiological implications by leverag- advances in device design and more sophisticated clinical ing the physical relationship between arterial pressure (AP) trial design, renal denervation has been proven effective,7 but and RBF. We test this method in both a chronic, surgical renal a clinically implementable way to validate the removal of the denervation rabbit model and an acute, functional renal dener- renal sympathetic nerves remains elusive. vation model in swine. We hypothesized that baroreflex-driven

Received April 19, 2020; first decision June 2, 2020; revision accepted July 20, 2020. From the Department of Anesthesiology (P.R.P., H.-J.W., A.M.S.), Department of Cellular and Integrative Physiology (I.H.Z., A.M.S., H-J.W.), and Division of Cardiovascular Medicine (Y.S.C.), University of Nebraska Medical Center, Omaha. The Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.120.15325. Correspondence to Alicia M. Schiller, University of Nebraska Medical Center, Omaha, NE. Email [email protected] © 2020 American Heart Association, Inc. Hypertension is available at https://www.ahajournals.org/journal/hyp DOI: 10.1161/HYPERTENSIONAHA.120.15325 1247 1248 Hypertension October 2020

rhythmic renal sympathetic nerve activity (RSNA) would give magnitude, and consistency of differences but not statistical signif- rise to rhythmic, baroreflex-driven, vasoconstrictive renovas- icance per se. These t statistics were calculated using a 2-tailed, cular modulation, termed renal sympathetic vasomotion. paired t test. Due to the high dimensionality of the occurrence data, statistical testing was performed with nonparametric cluster mass- based testing, which allows for a priori significance testing of non- Methods independent, multidimensional data while addressing the inherent All methods are further detailed in the Data Supplement. multiple comparisons problem.12 This is illustrated in Figure S2 and detailed further in the Data Supplement. Data and Materials Availability The data and source code from this study are available from the cor- Results responding author upon reasonable request. Rhythmic RSNA Rabbit Experiments Rabbits were instrumented with RSNA electrodes and AP Experiments were performed on adult male New Zealand White rab- telemeters to identify sympathetic rhythms likely to give bits. Both rabbit and pig experiments were reviewed and approved by rise to renal sympathetic vasomotion (Figure 1A). Short rep- our Institutional Animal Care and Use Committee and carried out in resentative tracings of AP and RSNA show rhythms occur- accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. ring approximately every 2 seconds in both signals as well as Five rabbits were instrumented with AP telemeters and RSNA the baroreflex control of RSNA, with low diastolic pressures electrodes under general anesthesia. After a 1-week recovery period followed by large bursts of RSNA. Five-minute sections of and acclimation to a procedure room, AP and RSNA were recorded. artifact-free AP and RSNA data underwent wavelet transfor- Another 10 rabbits were instrumented with AP telemeters and bilat- eral RBF probes and underwent unilateral surgical renal denervation mation to examine the rhythmic nature of AP and RSNA as under general anesthesia. After a 2-week recovery period and accli- well as cross-spectral analysis (Figure 1B). This cross-spectral mation to a procedure room, AP and bilateral RBF were recorded. At analysis reveals high coherence between AP and RSNA from the end of data collection, the nasopharyngeal reflex was elicited to 0.2 to 0.75 Hz. The cross-spectral phase shift demonstrates a validate unilateral denervation. In a separate experiment, the gangli- negative phase shift at this 0.2 to 0.75 Hz frequency range, onic blocker hexamethonium was administered to eliminate global autonomic sympathetic outflow. meaning that oscillations in RSNA follow oscillations in AP, which is consistent with baroreflex-driven control of RSNA. Swine Experiments Group data from all 5 rabbits are shown as occurrence histo- Experiments were carried out on 9 male domestic swine. Each pig grams in Figure 1C and 1D. The coherence occurrence his- was induced with tiletamine and zolazepam and intubated. Intrathecal togram shows that the AP-RSNA coherence is consistently Downloaded from http://ahajournals.org by on October 27, 2020 access at the caudal levels of the thoracic spine was obtained after dis- highest in the 0.2 to 0.75 Hz frequency range, indicating that section to the interspinous ligament, and an intrathecal catheter was advanced to the T10/T11 interspace under fluoroscopic guidance. The this is the frequency range with the strongest AP-RSNA rela- pig was maintained on a constant infusion of ketamine-midazolam tionship (Figure 1C). The phase shift occurrence histogram for the remainder of the experiment. Femoral venous and arterial also shows negative phase shift behavior between 0.2 and 0.75 access were obtained, and heparin was administered for thrombopro- Hz, indicating baroreflex control of RSNA in this frequency phylaxis. A pressure-flow velocity catheter was advanced to the renal range (Figure 1D). Baseline hemodynamics for these rabbits artery; renal AP-flow velocity data were acquired. Functional renal denervation was then performed by administering an intrathecal bolus are shown in Table S1. Taken together, these data demonstrate of ropivacaine, blocking both afferent and efferent neural transmis- strong, baroreflex-driven control of RSNA at the 0.2 to 0.75 sion around the level of the bolus and resulting in renal sympatholysis Hz frequency range in rabbits likely to give rise to renal sym- given the location of preganglionic renal sympathetic neurons at the pathetic vasomotion. T10-T11 level of the spinal cord.10,11 After waiting 15 minutes for the intrathecal ropivacaine to reach peak effect, renal AP-flow velocity data were again acquired for analysis. Novel Method for Vasomotion Assessment Previous attempts to investigate sympathetic control of the Data Analysis renal vasculature have relied on methods that assumed a rigid The relationship between AP and RSNA was analyzed using auto- renal artery and a time-invariant pressure-flow relationship, spectral and cross-spectral wavelet analysis over a wide physiolog- failing to account for the reality of pulsatile flow measured ical frequency range (0.03–2.5 Hz) to identify potentially important in the elastic renal artery that in turn perfuses a circulation sympathetic vasomotor frequencies in rabbits. In brief, AP-RSNA wavelet coherence and AP-RSNA wavelet phase shift were calcu- whose strong autoregulatory mechanisms dynamically control lated across each 5-minute recording, and group data was visualized the relationship between renal AP and flow. We developed a by creating occurrence histograms across time using 20 bins of equal novel method of assessing time-varying, rhythmic modulation size across the ranges [0, 1] and [−π, π] for coherence and phase of vascular resistance that accounts for the elastic nature of shift, respectively (illustrated graphically in Figure S1 in the Data Supplement). The novel vasomotion analysis method is introduced the artery in which flow is measured, and we then applied this alongside Figure 2 and further detailed in the Data Supplement. method to study renal sympathetic vasomotion. Pulsatile blood flow measured in an elastic artery can ei- Statistical Analysis ther travel into the capacitive artery or downstream across the Tabular data are displayed as mean±SEM. Statistical testing of arteriolar resistance (Figure 2A), and therefore, arterial blood simple measures was conducted with 2-tailed, paired t tests and, flow measurements depend on both arterial capacitance and where appropriate, repeated-measures–ANOVA with α=0.05. Group occurrence histograms are displayed as mean data for each arteriolar resistance. The renal circulation can then be mod- group with t statistics computed for each independent variable eled as a 2-element Windkessel with time-varying resistance pair (eg, frequency-admittance gain bin) to convey directionality, and capacitance, the electrical circuit abstraction of which is Ricci Pellegrino et al Sympathetic Vasomotion after Renal Denervation 1249

Figure 1. Rhythmic renal sympathetic nerve activity (RSNA) in conscious rabbits show frequency-dependent, baroreflex-driven coupling to arterial pressure (AP) rhythms. A, Rabbits were chronically instrumented with arterial pressure telemeters and renal sympathetic nerve electrodes. B, AP wavelet spectrum, RSNA wavelet spectrum, AP-RSNA coherence, and AP-RSNA phase shift from one representative rabbit demonstrate AP-RSNA coupling between 0.2 and 0.75 Hz with consistently negative phase shift. C, Group coherence occurrence histograms and (D) group phase shift occurrence histograms show high AP- RSNA coherence and negative AP-RSNA phase shift behavior across all 5 rabbits between 0.2 and 0.75 Hz. Downloaded from http://ahajournals.org by on October 27, 2020 shown in Figure 2B. As renal sympathetic adrenergic innerva- Thus, this assessment of vasomotion allows for a physio- tion is concentrated most densely on the renal arterioles,13 this logical interpretation of the vascular control of the circulation novel method focuses on control of renal vascular resistance. undergoing pressure-flow monitoring in real-time. Moreover, Thus, the resistive component of arterial blood flow was iso- certain physiological vascular control mechanisms operate lated by using the AP waveform to identify short intervals over primarily at certain frequencies, and thus frequency-guided which the mean arterial blood flow equaled the resistive flow vasomotion analysis allows one to focus on vascular control- (Figure 2C, see proof in the Data Supplement). Pressure, re- lers of interest with greater sensitivity and specificity. sistive flow, and resistance enjoy a simple, linear physical re- lationship. Thus, linear time-varying transfer function analysis Renal Sympathetic Vasomotion in Surgically of AP and resistive flow reveals modulation of vascular resist- Denervated Conscious Rabbits ance as a function of time and frequency (Figure 2D). This is To assess renal sympathetic vasomotion, rabbits underwent a powerful lens to view vascular control as the components of unilateral renal denervation and were instrumented with an AP the time-varying pressure-resistive flow transfer function each telemeter and bilateral RBF probes (Figure 3A), allowing for si- have a clear physiological interpretation. Admittance gain, the multaneous assessment of two kidneys exposed to the same sys- transfer function gain normalized by vascular conductance, temic milieu and perfusion pressure and differing solely by their provides an index of the active buffering of AP oscillations by sympathetic innervation. The novel vasomotion analysis method vascular resistance modulation. Phase shift provides informa- outlined above was used to calculate the relationship between tion about timing of oscillations in AP and blood flow, with AP and RBF of the innervated (INV) and surgically denervated negative phase shift indicating AP oscillations leading vas- (DNx) kidney at the 0.2 to 0.75 Hz frequency band previously cular resistance modulation, zero phase shift indicating pres- found to contain a strong baroreflex-driven RSNA rhythm in sure-flow synchrony, and positive phase shift indicating blood rabbits (Figure 1B). The admittance gain plots for the INV and flow oscillations leading resistance modulation. As a corol- DNx kidneys of one rabbit show a high prevalence of low admit- lary, phase shift also implies causality, with negative phase tance gain behavior in the INV kidney that is absent in the DNx shift consistent with baroreflex control of vascular resistance, kidney, consistent with the elimination of a vasoconstrictive con- zero phase shift consistent with passive Poiseuille flow, and trol mechanism by surgical renal denervation. Phase shift for the positive phase shift consistent with autoregulatory control of INV and DNx kidney of this rabbit reveals negative phase shift vascular resistance. Coherence quantifies how closely resis- behavior in the INV kidney which is replaced by zero phase shift tive RBF follows AP and is thereby inversely related to the behavior in the DNx kidney, consistent with the replacement of amount of active resistance modulation at a given frequency a baroreflex-mediated control mechanism by passive laminar over a particular time interval. flow after surgical renal denervation. Coherence for the INV and 1250 Hypertension October 2020 Downloaded from http://ahajournals.org by on October 27, 2020

Figure 2. Novel analysis method used to demonstrate renal sympathetic vasomotion. A, Anatomic model showing pulsatile blood flow from the heart through an elastic artery and resistive arterioles. B, Lumped circuit abstraction of anatomic model. C, Interval detection method used to extract the resistive

blood flow time series (Qres) from the raw blood flow time series using the arterial pressure (AP) signal.D , High-level overview of data analysis process by which AP and blood flow signals are converted into the 3 components of the pressure-resistive flow time-varying transfer function: admittance gain, phase shift, and coherence. PP indicates pulsatile pressure; and TVTF, time varying transfer function.

DNx kidneys shows more low-coherence behavior in the INV Statistical analysis of group data for coherence is shown in kidney, consistent with a greater amount of active vascular con- Figure 3E. Low-coherence behavior is more common in INV trol in the INV kidney than the DNx kidney at this important kidneys than DNx kidneys. Statistical testing revealed an ex- sympathetic frequency range. pansive, significant cluster of low-coherence behavior that was The results of nonparametric cluster mass-based statistical more common in INV kidneys, and a spatially concentrated, testing of group data for admittance gain behavior is shown in high-coherence cluster more common in DNx kidneys. As co- Figure 3C. As in the representative recording, negative admit- herence is inversely related to active vascular control, this dif- tance gain behavior is more prevalent in INV kidneys, manifest- ference is consistent with the elimination of an active vascular ing as negative t statistics in this region of the difference statistic control mechanism by renal denervation. map in Figure 3C, where DNx kidneys demonstrate more high Baseline hemodynamics for these rabbits are shown in admittance gain behavior, manifesting as positive t statistics in Table S2; note that there is no difference in mean RBF be- this region. Significance testing revealed both a statistically sig- tween INV and DNx kidneys. Wavelet autospectral param- nificant low admittance gain cluster that was more prevalent in eters did not differ between INV and DNx kidneys (Figure INV kidneys and a statistically significant passive admittance S3). Further vasomotion data can be found in Figure S4. The gain cluster that was more prevalent in DNx kidneys. This is completeness of surgical renal denervation was functionally consistent with the elimination of an active vasoconstrictive con- confirmed by evoking renal sympathetic vasoconstriction via trol mechanism and its replacement by passive transduction of the nasopharyngeal reflex (Figure S5). AP in this sympathetic frequency range. Statistical analysis of group data for phase shift is shown Effect of Ganglionic Blockade on Sympathetic in Figure 3D. Negative phase shift behavior is significantly Vasomotion in Rabbits more prevalent in INV kidneys; in DNx kidneys, pressure- We hypothesized that the observed vasomotion differences flow synchrony is significantly more common. This is con- between INV and DNx kidneys arose due to the baroreflex- sistent with the replacement of a baroreflex-driven vascular driven, rhythmic RSNA explored in Figure 1, and thus gan- control mechanism with passive pressure-flow transduction. glionic blockade with hexamethonium would eliminate these Ricci Pellegrino et al Sympathetic Vasomotion after Renal Denervation 1251

Figure 3. Renal vasomotion analysis reveals significant differences in unilaterally denervated, bilaterally instrumented conscious rabbits.A, Conscious, unilaterally denervated rabbit instrumented with an abdominal aortic pressure telemeter, a renal blood flow probe on the innervated (INV) kidney, and a renal blood flow probe on the surgically denervated (DNx) kidney.B , Representative tracings of admittance gain for an INV kidney, admittance gain for a DNx kidney, phase shift for an INV kidney, phase shift for a DNx kidney, coherence for an INV kidney, and coherence for a DNx kidney from one rabbit are displayed. Nonparametric cluster mass-based statistical analysis demonstrated significant differences in the C( ) admittance gain, (D) phase shift, and (E) coherence behavior for all 10 rabbits.

differences by blocking autonomic outflow. To test this hypo- ganglionic blockade at this low admittance gain region, in- thesis, rabbits were administered an intravenous bolus dose dicative that the effect of hexamethonium on admittance gain

Downloaded from http://ahajournals.org by on October 27, 2020 of hexamethonium known to eliminate RSNA while AP and depends on renal innervation. bilateral RBF were monitored.14 Figure 4D shows the group data for phase shift. Before Figure 4A shows representative tracings of AP and bilat- hexamethonium, negative phase shift behavior is signif- eral RBF for one rabbit treated with hexamethonium. icantly more prevalent in INV kidneys, whereas passive, Figure 4B demonstrates the effect of ganglionic blockade pressure-flow synchrony is significantly more prevalent in on the 3 components of the time-varying pressure-resistive flow DNx kidneys. Again, these differences were eliminated by transfer function of each kidney in this representative rabbit. As ganglionic blockade. Significance testing again identified in Figure 3, the INV kidney exhibits more negative admittance a statistically significant interaction between innervation gain behavior than the DNx kidney in the baseline state. This status and ganglionic blockade at these areas of baseline difference is eliminated after administration of hexamethonium, difference in phase shift between INV and DNx kidneys, resulting in nearly identical admittance gain plots between the indicating that the effect of hexamethonium on phase shift INV and DNx kidneys. Mirroring Figure 3, the INV kidney depends on renal innervation. shows more negative phase shift vasomotion, whereas the DNx The coherence group data shows a similar phenomenon kidney shows pressure-flow synchrony. Ganglionic blockade (Figure 4E). In the setting of normal ganglionic transmis- abrogates this difference, resulting in strikingly similar phase sion, low-coherence behavior is significantly more preva- shift plots for the INV and DNx kidney after hexamethonium lent in INV kidneys and high coherence is more prevalent administration. Where the DNx kidney is characterized by pas- in DNx kidneys, and this difference is eliminated after ad- sive high-coherence behavior in the baseline state, the INV ministration of hexamethonium. Figure 4E shows a statisti- kidney exhibits low-coherence behavior consistent with active cally significant interaction between innervation status and vasomotion. This difference is again eliminated by blocking au- hexamethonium. tonomic ganglionic transmission. Note that movement artifacts In summary, administration of hexamethonium eliminates are characterized by aberrant vasomotion, independent of inner- the difference between INV and DNx kidneys, consistent with vation or ganglionic blockade. the hypothesis that the RSNA rhythms observed in Figure 1 Group data (n=9) for the hexamethonium experiments drive the observed differences in vasomotion between INV are shown in Figure 4C through 4E, with Figure 4C focusing and DNx kidneys. on admittance gain. In the baseline state, INV kidneys ex- Group hemodynamics are shown in Figure S6, which hibit significantly more negative admittance gain behavior, demonstrates that hexamethonium significantly reduces AP whereas DNx kidneys exhibit more positive admittance gain and raises heart rate but does not significantly affect mean behavior; the differences between INV and DNx kidneys are RBF. Additional autospectral and cross-spectral data are eliminated by hexamethonium. Significance testing identified shown in Figures S7 through S9; note that wavelet analysis a statistically significant interaction between innervation and shows that hexamethonium treatment decreases the spectral 1252 Hypertension October 2020

Figure 4. Ganglionic blockade eliminates differences between innervated (INV) and surgically denervated (DNx) kidneys in unilaterally denervated rabbits. A, Tracings of arterial pressure, renal blood flow to the INV kidney, and renal blood flow to the DNx kidney before (Baseline) and after hexamethonium (Hex) administration show the hemodynamic effects of hexamethonium administration in one representative rabbit. B, Representative tracings of admittance gain of the INV kidney before hexamethonium, admittance gain of the DNx kidney after hexamethonium, admittance gain of the DNx kidney before hexamethonium, admittance gain of the DNx kidney after hexamethonium, phase shift of the INV kidney before hexamethonium, phase shift of the INV kidney after hexamethonium, phase shift of the DNx kidney before hexamethonium, phase shift of the INV kidney after hexamethonium, coherence of the INV kidney before hexamethonium, coherence of the INV kidney after hexamethonium, coherence of the DNx kidney before hexamethonium, and coherence of the INV kidney after hexamethonium from this rabbit are displayed. C, Statistical testing of admittance gain behavior from all 9 rabbits shows significant INV-DNx differences in the baseline state that are eliminated after hexamethonium treatment and a significant interaction between innervation and ganglionic blockade. D, Statistical testing of phase shift behavior from all rabbits shows significant INV-DNx differences in the baseline state that are eliminated after hexamethonium treatment and a significant interaction between innervation and ganglionic blockade.E. Statistical testing of coherence behavior from all

Downloaded from http://ahajournals.org by on October 27, 2020 rabbits shows significant INV-DNx differences in the baseline state that are eliminated after hexamethonium treatment and a significant interaction between innervation and ganglionic blockade. NS indicates non-significant interaction.

energy of the AP and bilateral RBF signals and that hexame- function analysis from this representative pig study is shown thonium greatly impacts the time-varying transfer function in Figure 5C. Admittance gain plots for this experiment reveal of both INV and DNx kidneys. a higher prevalence of low admittance gain behavior in the baseline state compared with the postropivacaine state, con- Renal Sympathetic Vasomotion in Anesthetized sistent with the elimination of active vasoconstriction by func- Swine Undergoing Acute Functional Denervation tional renal denervation. Phase shift plots for this experiment Although the results of these rabbit studies establish a also reveal a reduction in negative phase shift behavior after theoretical basis for renal sympathetic vasomotion, this administration of ropivacaine, indicating a reduction in baro- technique needed to be validated with clinical technology reflex-driven renal vascular modulation. The negative phase in a more translational model. Thus, we tested this novel shift behavior is replaced by positive phase shift behavior, con- method in a swine model of acute renal denervation. sistent with active autoregulatory vascular control. Coherence Anesthetized swine were instrumented with an intrathecal is not obviously affected by ropivacaine administration in this catheter at the T10/T11 level (Movies S1 and S2), and an representative experiment, indicating persistent active vasomo- intravascular pressure-flow velocity wire was advanced tion in this frequency range despite renal sympatholysis. to the renal artery (Movie S3). Although recording renal Figure 5D through 5F show occurrence group data for all AP and blood flow velocity, functional renal denervation 9 pigs. As in the representative experiment, low admittance was achieved by injection of an intrathecal bolus of ropi- vacaine, blocking preganglionic renal sympathetic nerve gain behavior is more prevalent in the baseline state and high traffic (Figure 5A). admittance gain behavior is more prevalent after renal sympa- The vasomotion analysis method was focused on the sym- tholysis (Figure 5D). Again, this is consistent with the abro- pathetic range of 0.03 to 0.10 Hz in swine.15 Of note, this fre- gation of a vasoconstrictive controller operating at this known quency range is more analogous to that of man (0.04–0.15 Hz) sympathetic vasomotor frequency. and presents a clinically relevant challenge as it overlaps with Phase shift group data in Figure 5E shows that negative the operating frequencies of renal autoregulatory mechanisms phase shift behavior is significantly more prevalent before ad- (0.02–0.25 Hz). ministration of intrathecal ropivacaine and, after ropivacaine, Figure 5B shows representative tracings of pulsatile AP and positive phase shift behavior is significantly more prevalent. renal AP and blood flow velocity over the course of a single This is indicative of the replacement of baroreflex-driven vas- experiment. Time-varying pressure-resistive flow transfer cular control by autoregulatory control. Ricci Pellegrino et al Sympathetic Vasomotion after Renal Denervation 1253

Figure 5. Renal vasomotion analysis reveals significant differences in anesthetized pigs undergoing functional renal sympathetic denervation.A , Anesthetized pigs underwent placement of a thoracic intrathecal catheter and renal arterial pressure-flow wire.B , Arterial pressure and renal blood flow velocity from one representative pig experiment are shown before (baseline) and after a bolus of intrathecal ropivacaine (Ropi), which blocks renal sympathetic outflow.C , Representative tracings of admittance gain in the baseline and post-Ropi states, phase shift in the baseline and post-Ropi states, and coherence in the baseline and postropivacaine states are shown for this representative pig. Nonparametric cluster mass-based statistical analysis shows significant differences in the D( ) admittance gain and (E) phase shift but not (F) coherence behavior for all 9 swine. NS indicates non-significant interaction.

Downloaded from http://ahajournals.org by on October 27, 2020 Figure 5F shows group data for coherence for the swine sympathetic vasomotion was quantified as the cumulative study. Unlike surgical renal denervation in rabbits, functional occurrence of vasoconstrictive, baroreflex-mediated renal renal denervation in swine does not significantly affect coher- vasomotion arising at sympathetic vasomotor frequencies. ence, a marker of the amount of active renovascular control. Renal sympathetic vasomotion was significantly decreased in Baseline hemodynamics for swine are shown in Table S3, the surgically denervated kidney of all ten rabbits (Figure 6A). intrathecal ropivacaine did not significantly affect AP, heart Intrathecal ropivacaine also significantly decreased quantifi- rate, or renal AP and blood flow velocity. Autospectral data able renal sympathetic vasomotion in swine (Figure 6B). are shown in Figure S10; note that AP spectral power in this Thus, this simplistic, a priori quantification method reflects sympathetic frequency range is reduced after intrathecal ropi- changes in renal sympathetic outflow in 2 distinct preclinical vacaine administration, consistent with effective sympatholy- models of renal denervation and could potentially be general- sis. Additional renal vasomotion data are found in Figure S11. ized for intraprocedural clinical use.

Renal Denervation Decreases Quantifiable Renal Discussion Sympathetic Vasomotion Although, in general, the results from the rabbit and swine The above multidimensional renal vasomotion data, al- models of renal denervation are largely corroborative, a few though identifying a renal sympathetic vascular control sig- key differences merit further discussion. The autoregula- nature in both rabbits and swine, are not readily interpretable tory control mechanisms of the kidney operate between 0.02 for intraprocedural decision-making. To address this, renal and 0.25 Hz in all species in which they have been studied9;

A B Renal Sympathetic Vasomotion Renal Sympathetic Vasomotion Figure 6. Quantification of renal sympathetic 30 ) 80 P < 0.01 vasomotion. A, Unilateral surgical renal %)

P < 0.01 (% denervation decreases the cumulative e( occurrence of negative admittance gain, 60 20 negative phase shift behavior at sympathetic vasomotor frequencies in rabbits. B, 40 Occurrenc Occurrence Functional renal denervation in swine 10 decreases the cumulative occurrence of 20 negative admittance gain, negative phase shift behavior at sympathetic vasomotor frequencies in swine. DNx indicates surgically Cumulative 0 Cumulative 0 INV DNx BaselineRopi denervated; and INV, innervated. 1254 Hypertension October 2020

conversely, the sympathetic rhythms that give rise to Mayer constant) pressure-flow relationship, which is incompatible waves occur at species-specific frequencies. In rabbits, the 0.2 with the known importance of renal autoregulation, and did to 0.75 Hz RSNA rhythm that we identified operates in a dis- not account for the elastic nature of the renal artery. This may tinct frequency range with little overlap of that of the auto- explain why previous studies investigating the importance of regulatory mechanisms. Conversely, in pigs and humans, the sympathetic control of the renal vasculature have arrived at sympathetic rhythms operate entirely in the frequency range of such heterogenous conclusions.16–19 That said, all vascular the autoregulatory controllers. Thus, although renal denerva- trees are known to exhibit viscoelastic and inductive proper- tion in both rabbits and swine reduced the amount of negative ties that are absent in our model. The renal circulation specif- phase shift behavior, consistent with a reduction in baroreflex ically is even more complex, boasting a dual series arteriolar control, in rabbits, this behavior was replaced by pressure-flow network with filtration function. synchrony, consistent with a passive pressure-flow relation- Our study has several limitations. For example, we are ship, whereas in pigs it was replaced by positive phase shift be- unable to quantify the density of the intrathecal ropivacaine havior, consistent with active autoregulatory control. Similarly, blockade, but we are certain that it is not as complete as the coherence, which is inversely related to the amount of active surgical denervation procedure. As early studies in humans vascular control, was increased by renal denervation in rab- indicate that full renal denervation was never achieved with bits but not pigs. This reflects the fact that active autoregula- the early endovascular radiofrequency ablation devices,20 this tory vasomotion continues in pigs at the frequency of interest; may render the study more clinically relevant. Additionally, whereas in rabbits there is no other vascular control mechanism we did not simultaneously measure RBF and RSNA in the that operates in this 0.2 to 0.75 Hz frequency range. same kidney, which would have been a very elegant, albeit The most immediate potential implications of this tech- technically challenging, model to further characterize renal nology pertain to the field of therapeutic renal denervation sympathetic vasomotion. Furthermore, this study was per- where interventionists have no way of validating the success of formed in healthy animals and leaves unaddressed the added a procedure. Renal sympathetic vasomotion monitoring could complexity of pathophysiology of human cardiovascular di- be performed clinically using the same approach employed for sease and its potential impact on renovascular control. swine with real-time assessment of renal sympathetic vasomo- To be translated to the clinic, similar preclinical studies tion used to provide intraprocedural feedback for clinicians. in which renal denervation is performed with minimally in- Measurement of renal AP-flow might be performed to measure vasive, state-of-the-art clinical devices and compared against baseline renal sympathetic vasomotion and then again after a gold-standard surgical denervation would provide impor-

Downloaded from http://ahajournals.org by on October 27, 2020 catheter-based renal denervation to verify effective dener- tant insights while avoiding the circular logic of validating a vation or, if the drop in renal sympathetic vasomotion were new diagnostic technology with an unverifiable intervention. unsatisfactory, prompt an additional ablation. Such feedback Moreover, acquisition of renal AP and flow velocity signals in could improve the efficacy, safety, and usability of this antihy- patients undergoing renal denervation procedures should be pertensive therapy. Moreover, as renal sympathetic vasomotion designed to further address issues about feasibility and safety can be characterized noninvasively (eg, using transabdominal in human hypertensive subjects. Doppler to measure RBF velocity and a volume-clamp device to measure AP), this technology could also be used to identify Perspectives potential therapy responders before the procedure and to mon- In this article, we identified a baroreflex-mediated RSNA itor for reinnervation after a successful procedure. rhythm that we hypothesized would give rise to rhythmic Beyond this, our approach has applications to other pro- modulation of the renal vasculature, termed renal sympathetic cesses involving the sympathetic nervous system. As the sym- vasomotion. We developed a novel method for characterizing pathetic nervous system is the endogenous hemodynamic rhythmic vascular modulation using measurements of simul- monitoring system and first-responder, the ability to detect taneous arterial blood pressure and RBF, and we tested it in acute subclinical activation of the sympathetic nervous system 3 different models of renal denervation. First, we tested the may improve our ability to identify patients at risk for hemo- technique in conscious rabbits that underwent unilateral sur- dynamic compromise and to intervene earlier. A more clin- gical renal denervation and instrumentation with bilateral ically viable method may also help improve the diagnosis, RBF probes. In these rabbits, INV kidneys exhibited more treatment, and understanding of chronic diseases like dysauto- negative admittance, negative phase shift, and low-coherence nomia, chronic heart failure, hypertension, and chronic kidney behavior compared with their DNx counterparts, consistent disease. Similarly, this method provides a powerful lens for with the presence of vasoconstrictive, baroreflex-mediated, assessing active vascular control of any origin. This approach active vasomotion caused by rhythmic RSNA. Next, gan- could be used to characterize autoregulatory control on a glionic blockade with hexamethonium was used to acutely patient-by-patient basis and to create technologies designed to eliminate global sympathetic nerve activity, and this also optimize the perfusion pressure of vital organs. eliminated the differences between INV and DNx kidneys, This method makes 2 assumptions: (1) that perfusion further corroborating the hypothesis that this vasomotion was pressure can adequately be approximated by AP and (2) the the result of rhythmic RSNA. Finally, this novel method was renal pressure-flow relationship can acceptably be described tested in anesthetized pigs that underwent acute functional by a time-varying, 2-component Windkessel lumped model. renal sympathetic denervation with thoracic intrathecal rop- This is a significant advancement over previous analysis ivacaine while renal artery pressure and flow velocity were of the renal circulation that assumed a time-invariant (ie, monitored with a clinically approved intravascular catheter. Ricci Pellegrino et al Sympathetic Vasomotion after Renal Denervation 1255

Again, functional renal denervation reduced the amount of 5. Kandzari DE, Bhatt DL, Brar S, Devireddy CM, Esler M, Fahy M, negative admittance gain and negative phase shift behavior, Flack JM, Katzen BT, Lea J, Lee DP, et al. Predictors of blood pressure re- sponse in the SYMPLICITY HTN-3 trial. Eur Heart J. 2015;36:219–227. consistent with the abrogation of a vasoconstrictive, barore- doi: 10.1093/eurheartj/ehu441 flex-mediated renovascular control mechanism. Additionally, 6. Esler M. Illusions of truths in the Symplicity HTN-3 trial: generic design quantifying renal sympathetic vasomotion as the amount of strengths but neuroscience failings. J Am Soc Hypertens. 2014;8:593–598. doi: 10.1016/j.jash.2014.06.001 vasoconstrictive, baroreflex-mediated renal vasomotion re- 7. 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Pellegrino PR, Schiller AM, Haack KK, Zucker IH. Central angiotensin- P.R. Pellegrino has received honoraria and travel expenses from II increases blood pressure and sympathetic outflow via Rho Kinase ac- Medtronic plc. P.R. Pellegrino, I.H. Zucker, Y.S. Chatzizisis, H.-j. tivation in conscious rabbits. Hypertension. 2016;68:1271–1280. doi: Wang, and A.M. Schiller have received a grant from Medtronic plc for 10.1161/HYPERTENSIONAHA.116.07792 further experiments based on this work. P.R. Pellegrino, I.H. Zucker, 15. von Borell E, Langbein J, Després G, Hansen S, Leterrier C, Y.S. Chatzizisis, H.-j. Wang, and A.M. Schiller have submitted a Marchant-Forde J, Marchant-Forde R, Minero M, Mohr E, Prunier A, et al. Heart rate variability as a measure of autonomic regulation of car- relevant provisional patent application (Docket No. 18060P, Serial diac activity for assessing stress and welfare in farm animals – a review. No. PCT/US19/19110), and P.R. Pellegrino, I.H. Zucker, and A.M. 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Novelty and Significance What Is New? Summary • We developed a novel technique called sympathetic vasomotion, a We introduced sympathetic vasomotion and demonstrated its va- marker of sympathetic control of the renal vasculature that could be used lidity both in a highly controlled rabbit model and a clinically rel- as a clinical end point for evaluating effective renal denervation. evant swine model of renal denervation. This technique could What Is Relevant? provide intraprocedural feedback for interventionists performing • Renal denervation is a paradigm-shifting antihypertensive intervention therapeutic renal denervation. that has struggled to gain traction due to the inability to validate pro- cedural success; sympathetic vasomotion could address this problem.